The renin-angiotensin system (RAS) is one of the most important regulatory mechanisms of body fluid and electrolyte homeostasis and blood pressure maintenance. The juxtaglomerular apparatus (JGA) is a key anatomical site of RAS where renin, the rate-limiting step of RAS, and its precursor prorenin are synthesized and released in a highly regulated fashion. One of the recent exciting topics in RAS research is the discovery of the (pro)renin receptor [(P)RR] challenging the existing simplistic RAS model (angiotensinogen, renin, converting enzyme, angiotensin (Ang) peptides and receptors). (Pro)renin binding by the receptor not only causes non- proteolytic activation of the enzyme and generation of AngI, but also triggers AngII-independent intracellular signaling. A functional role for prorenin was suspected long ago since elevated plasma prorenin is a well-recognized predictor of microvascular complications in cardiovascular disease and diabetes. Prorenin and its receptor have become new elements of RAS, hot players and therapeutic targets in disease. During the past grant cycle we successfully characterized important JGA functions, the tubuloglomerular feedback (TGF) and renin release mechanisms and their important constituents including the ATP and connexin-mediated calcium wave. Also, we pioneered a unique multi-photon imaging approach to directly and quantitatively visualize the intact kidney, monitor the basic parameters of kidney function in vivo including (pro)renin content, release and tissue activity. Our overall hypothesis is that the (P)RR, localized in the basolateral membrane of macula densa (MD) cells, and its downstream signaling is an important and novel modulator of MD and JGA functions. More precisely, we hypothesize that the (P)RR constitutes a short-loop positive feedback stimulating renin release from adjacent JG cells which includes activation of MD MAP kinases and the classic PGE2 synthetic and release machinery. We also hypothesize that another novel, but inhibitory JGA mechanism, a connexin and ATP-mediated vascular calcium signal helps to balance renin synthesis and release. Aim 1 will test for the expression, regulation, and signaling of the (P)RR in macula densa cells using molecular techniques. Aim 2 will establish the role of macula densa (P)RR in JGA function using multi-photon fluorescence imaging of the intact kidney in vivo or freshly dissected, microperfused JGA preparations in vitro and a number of transgenic approaches and animal models. Aim 3 will identify the mechanism and importance of Cx45/ATP-mediated inhibition of renin release using multi-photon imaging. These original and novel studies are expected to provide clinically important information that can be used to develop new drugs and therapeutic approaches for the better treatment of cardiovascular and kidney diseases. PUBLIC HEALTH RELEVANCE Hypertension is a major risk factor for cardiovascular diseases, affecting about 25% of the population. The kidney is central to maintenance of normal blood pressure and to the pathogenesis of hypertension because of its dominant role in the regulation of salt and water balance. In this proposal we will directly visualize novel mechanisms in the kidney using a state-of- the-art imaging technology which will allow us to discover how the kidney operates in real time to control blood volume and pressure.